242 Am. J. Trop. Med. Hyg., 65(3), 2001, pp. 242–251 Copyright  2001 by The American Society of Tropical Medicine and Hygiene PHYLOGENETIC ANALYSIS OF JAPANESE ENCEPHALITIS VIRUS: ENVELOPE GENE BASED ANALYSIS REVEALS A FIFTH GENOTYPE, GEOGRAPHIC CLUSTERING, AND MULTIPLE INTRODUCTIONS OF THE VIRUS INTO THE INDIAN SUBCONTINENT PRADEEP D. UCHIL AND VIJAYA SATCHIDANANDAM Department of Microbiology And Cell Biology, Indian Institute of Science, Bangalore, India Abstract. We report the analysis of the complete nucleotide sequence for the Indian isolate (P20778; Genbank Accession number AF080251) of Japanese encephalitis virus (JEV). The phylogenetic tree topology obtained using thirteen complete genome sequences of JEV was reproduced with the envelope, NS1, NS3, and NS5 genes and revealed extensive divergence between the two Indian strains included. A more exhaustive analysis of JEV evolution using 107 envelope sequences available for isolates from different geographic locations worldwide revealed five distinct genotypes of JEV, displaying a minimum nucleotide divergence of 7% with high bootstrap support values. The tree also revealed overall clustering of strains based on geographic location, as well as multiple introductions of JEV into the Indian subcontinent. Nonsynonymous nucleotide divergence rates of the envelope gene estimated that the ancestor common to all JEV genotypes arose within the last three hundred years. INTRODUCTION Japanese encephalitis virus (JEV) is the cause of the most prevalent viral encephalitis of man in terms of morbidity and mortality. 1 Approximately 50,000 human cases of JE occur annually in Asia. 2 Japanese encephalitis virus belongs to the family Flaviviridae, whose members include several patho- gens of humans and animals. In India, epidemics of JEV have been reported since the mid-1950s, and the virus is now endemic in most parts of the country. The genomes of fla- viviruses comprise single-stranded RNA of positive polarity approximately 11,000 nucleotides (nt) in length. The struc- tural proteins: capsid, premembrane, and envelope, are en- coded in the 5 third of the genome and are followed by the genes specifying the seven nonstructural proteins. Japanese encephalitis virus is one of the recently diverged members of the family Flaviviridae, and most JEV isolates are estimated to have evolved over the last 130 years. 3 The study of flavivirus phylogeny has primarily utilized the structural glycoprotein envelope sequences. 4 These, in ad- dition to serological studies, 5 have elegantly shown differ- ences in evolutionary dynamics of the tick- and mosquito- borne subgroups of this genus. Phylogenetic analysis of se- quences from the nonstructural RNA dependent RNA poly- merase (NS5) gene of mosquito- and tick-borne flaviviruses 6,7 have also yielded tree topologies similar to that obtained with envelope sequences. More recently, NS3, the full-length genome sequence, and NS5 were used with equal success to classify the nonvector-borne and arthropod-borne flaviviruses. 8 To study JEV evolution, on the other hand, a stretch of 240 or 198 nt from the premembrane (prM) region have been predominantly used. 9–13 These studies have classified JEV strains into four distinct genotypic groups. Another tree building exercise using a limited number of twenty JEV en- velope sequences identified four clusters which however did not match the four genotypes mentioned above and also ‘‘did not correspond to geographic origin, isolation host, or viru- lence’’. 14 Yet another analysis which compared six full- length JEV sequences suggested that the Indian strain GP78 (Accession number AF075723) may be related to the Chi- nese SA-14 isolate. 15 Most of these studies utilized uncor- rected p-distances (proportion of nucleotide or amino acid sites at which the two sequences compared are different) for building phylogenetic trees. Our objective was three-fold. Firstly, to study the evolu- tion of JEV, which required identification of good phyloge- netic markers to replace the 240 nt stretch used previously (see below). We have determined and analyzed the complete nucleotide sequence for an Indian isolate P20778 of JEV and carried out phylogenetic analysis of this and twelve other independently isolated full-length JEV sequences available in the database at the nucleotide and amino acid sequence level. The ten individual genes were separately analyzed us- ing the nucleotide sequences to assess their utility as phy- logenetic markers. Our second objective was to analyze the extensive genetic diversity of Indian JEV strains 9,16 and to see if strain varia- tion of JEV from different parts of the globe could be linked to their geographic origin. The literature on this topic is con- flicting. 9,17 Therefore, an exhaustive tree building exercise was undertaken with all 107 unique envelope sequences available in the database of JEV strains from geographically diverse regions of Asia. This revealed the hitherto unrec- ognized fifth genotype of JEV. Thirdly, we determined the date of the JEV ancestor com- mon to all 5 E-gene based genotypes we have identified. This study thus represents an up-to-date, rigorous, and in- depth phylogenetic analysis of JEV isolates. MATERIALS AND METHODS Cells and viruses. The Aedes albopictus cell line C6/36 (obtained from NCCS, Pune, India) was maintained in Ea- gle’s minimum essential medium (MEM) with 10% fetal bo- vine serum (FBS), and was used for growth of virus. Japa- nese encephalitis virus strain P20778 from Vellore 18 was used for sequence determination reported here. Confluent monolayers of C6/36 were infected with virus at a multi- plicity of infection (m.o.i.) of 0.1. Medium containing virus was harvested twice, at day four and again at day eight post- infection (p.i.). Virus titer was determined on monolayers of